Section III: Sequencing Troubleshooting Quick Reference Table
Sequence Analysis Troubleshooting Guide
29
Only signal of the
Unincorporated
dyes observed with a
normal Current
Profile
Failed Enzymatic Reaction
Check T
m
of primer and work out the
correct annealing temperature
Incorrect primer
Check sequenced T
m
of primer and
work out the correct annealing
temperature
Incorrect annealing
temperature
Check T
m
of primer and work out the
correct annealing temperature
Did not add one of the
reagents in the chemistry
setup
Check that all reagents were added
Malfunctioning Thermal
Cycler
Check the thermal cycler program for
correct program and ramping
Check Thermal Cycler
No signal associated
with bad Current
profile
See explanation on erratic or
crashed current profiles above.
Identical Erratic or
Crashed Current
profile in:
All eight capillaries
of the array
Air bubbles coming from the
Manifold end
Do 2 manual Manifold Purges and 2
manual Gel Fills from the Direct
Control section of the Run module and
rerun the samples (Note: use a fresh
buffer plate)
Manifold area dirty thus not
allowing for the manifold end
of the array to be sealed
correctly
Clean Manifold area of instrument
The current can be affected in
all eight capillaries if not
enough buffer is added to the
buffer plate
Fill buffer plate well at least ¾ full
One capillary
Air Bubbles in the Sample
Well
Check sample plate for air bubble
before loading onto CEQ
Too much DNA template
(These peaks do not tend to be
as sharp as those caused by air
bubbles)
Reduce the amount of template in cycle
sequencing of template in cycle
sequencing reaction
Preheat super-coiled template DNA
prior to cycle sequencing
PROBLEM
RAW DATA
SIGNAL /
CURRENT
PROFILE
POTENTIAL SOURCE OF
PROBLEM
POSSIBLE SOLUTIONS
